Depolymerization of post-consumer PET bottles with engineered cutinase 1 from<i>Thermobifida cellulosilytica</i>

Zhang, Zixuan; Huang, Shiming; Cai, Di; Shao, Chaofeng; Zhang, Changwei; Zhou, Jinyun; Cui, Ziheng; He, Tao; Chen, Changjing; Chen, Biqiang; Tan, Tianwei

doi:10.1039/d2gc01834a

Cited by 20 publications

(19 citation statements)

References 50 publications

(78 reference statements)

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“…However, high-level candidate degraders with the ability to produce degradation products such as mono/bis(2-hydroxyethyl) terephthalate (MHET/BHET) and terephthalate (TPA) on an industrial scale are limited and need further approaches to design efficient PET hydrolases as well as to achieve ultra-high-throughput screening from novel resources (Wei et al 2022 ). Among already known candidates, cutinases from thermophilic actinomycetes ( T. fusca , T. cellulosilytica and Saccharomonospora viridis ) (Wei et al 2019 ; Zhang et al 2022 ; Emori et al 2020 ), a leaf-branch compost metagenome-derived cutinase (LCC) (most probably derived from Chloroflexi species; Xi et al 2021 ) (Tournier et al 2020 ; Zeng et al 2022 ) and a thermophilic fungus cutinase from Humicola insolens (commercialized) (Castro et al 2019 ) have been reported to possess a thermostability at more than 70 °C and to accumulate high millimolar level of degradation products. Tournier et al ( 2020 ) reported an efficient process for PET recycling, based on two strategies using amorphized and particulate PET with engineered variants of the LCC.…”

Section: Introductionmentioning

confidence: 99%

Efficient depolymerization of polyethylene terephthalate (PET) and polyethylene furanoate by engineered PET hydrolase Cut190

et al. 2022

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The enzymatic recycling of polyethylene terephthalate (PET) can be a promising approach to tackle the problem of plastic waste. The thermostability and activity of PET-hydrolyzing enzymes are still insufficient for practical application. Pretreatment of PET waste is needed for bio-recycling. Here, we analyzed the degradation of PET films, packages, and bottles using the newly engineered cutinase Cut190. Using gel permeation chromatography and high-performance liquid chromatography, the degradation of PET films by the Cut190 variant was shown to proceed via a repeating two-step hydrolysis process; initial endo-type scission of a surface polymer chain, followed by exo-type hydrolysis to produce mono/bis(2-hydroxyethyl) terephthalate and terephthalate from the ends of fragmented polymer molecules. Amorphous PET powders were degraded more than twofold higher than amorphous PET film with the same weight. Moreover, homogenization of post-consumer PET products, such as packages and bottles, increased their degradability, indicating the importance of surface area for the enzymatic hydrolysis of PET. In addition, it was required to maintain an alkaline pH to enable continuous enzymatic hydrolysis, by increasing the buffer concentration (HEPES, pH 9.0) depending on the level of the acidic products formed. The cationic surfactant dodecyltrimethylammonium chloride promoted PET degradation via adsorption on the PET surface and binding to the anionic surface of the Cut190 variant. The Cut190 variant also hydrolyzed polyethylene furanoate. Using the best performing Cut190 variant (L136F/Q138A/S226P/R228S/D250C-E296C/Q123H/N202H/K305del/L306del/N307del) and amorphous PET powders, more than 90 mM degradation products were obtained in 3 days and approximately 80 mM in 1 day. Graphical Abstract

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Section: Introductionmentioning

confidence: 99%

Efficient depolymerization of polyethylene terephthalate (PET) and polyethylene furanoate by engineered PET hydrolase Cut190

et al. 2022

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“…With the crystal structures of several PHEs elucidated, structure‐based rational designs of PHEs for improved hydrolytic activity and thermostability have been intensively researched and made great progress (Table 1). Reshaping the substrate‐binding pocket by exploring key residues around the binding cleft, [26,29] optimizing electrostatic charge of protein surface, [19a,30] introducing additional interactions (disulfide bonds, [19a,29a,f,30–31] salt bridges, [32] hydrogen bonds, [33] hydrophobic [33–34] and aromatic interactions [35] ), and increasing proline which has a rigid cyclic structure on the side chain [34] for higher thermostability are commonly used strategies. For example, Tournier et al [29a] .…”

Section: Protein Engineering Of Pet Hydrolytic Enzymesmentioning

confidence: 99%

Recent Advances and Challenges in Enzymatic Depolymerization and Recycling of PET Wastes

Shi,

Zhu

2023

ChemBioChem

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Poly (ethylene terephthalate) (PET) is one of the most commonly used plastics in daily life and various industries. Enzymatic depolymerization and recycling of post‐consumer PET (pc‐PET) provides a promising strategy for the sustainable circular economy of polymers. Great protein engineering efforts have been devoted to improving the depolymerization performance of PET hydrolases. In this review, we firstly discuss the mechanisms and challenges of enzymatic PET depolymerization. Subsequently, we summarize the state‐of‐the‐art engineering of PET hydrolases including rational design, machine learning and directed evolution for improved depolymerization performance, and highlight the advances in screening methods of PET hydrolases. We further discuss several factors that affect the enzymatic depolymerization efficiency. We conclude with our perspective on the opportunities and challenges in bio‐recycling and bio‐upcycling of PET wastes.

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“…15). Second, the surfactant addition and dosage were tested as surfactants have been proven to be an efficient method for accelerating the PET hydrolysis process [39][40][41] . The enzyme concentration was optimized last considering the optimal enzyme loading was correlated with the surfactant addition (Supplementary Fig.…”

Section: The Depolymerization Of Untreated Post-consumer Pet (Pc-pet)...mentioning

confidence: 99%

Whether the wobbling W156 is a pre-requisite for efficient PET biodegradation by IsPETase

Yin

Zhang

et al. 2023

Preprint

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IsPETase displayed great potential for mitigating PET contamination under moderate temperatures attributed to its specific high activity towards PET, while rapid activity loss limited its commercial viability. Herein, a thermostable S92P/D157A variant (Tm=70.8 °C) was constructed, enabling a 109.3-fold increase in products released from amorphous PET depolymerization at 40 °C. Further depolymerization of untreated post-consumer PET in a 360-times expanded reaction system obtained 20.09 mM products (95.0% TPA). Crystal structure characterized the “wobbling” W156 residue fixed at substrate-binding conformation in S92P/D157A variant, which stabilized the enzyme structure and could provide steady interaction with the substrate. Further MD simulations cement the importance of conformation selection and the contribution of W156. The different structure-function relationship observed raised the question of “whether the wobbling W156 is a pre-requisite for efficient PET biodegradation”. Especially, the S92P/D157A variant could serve as a starting point for further engineering and a potential candidate for industrial PET depolymerization processes.

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Depolymerization of post-consumer PET bottles with engineered cutinase 1 fromThermobifida cellulosilytica

Abstract: Bio-recycling of plastic waste is a promising solution to plastic pollution. As one of the most abundant plastic wastes, polyethylene terephthalate (PET) can be degraded by carboxylic ester hydrolases (EC...

Cited by 20 publications

References 50 publications

Efficient depolymerization of polyethylene terephthalate (PET) and polyethylene furanoate by engineered PET hydrolase Cut190

Efficient depolymerization of polyethylene terephthalate (PET) and polyethylene furanoate by engineered PET hydrolase Cut190

Recent Advances and Challenges in Enzymatic Depolymerization and Recycling of PET Wastes

Whether the wobbling W156 is a pre-requisite for efficient PET biodegradation by IsPETase

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